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<div>Hi John,</div>
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<div>just some further suggestions.</div>
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<div>What is your cooperation partner interested in - a) size DISTRIBUTION, b) molar mass (accuracy and precisision) or c) conformation of the DNA?</div>
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<div>Depending on the answers, you should select your experiment.</div>
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<div>ad a) SV, as fast as possible, preferably with interference - do you have scattering at 260 nm at the relevant concentrations? Almost no diffusion, so ls-g(s). Mw from scaling relations, if available. Requires prior knowledge or assumptions, though.</div>
<div>ad b) SE is a possibility for such large complexes and can give you very precise molar masses, to feed back any scaling relations there might be (Steve Harding can tell you all and more about that). Use 50 microL of filling volume (12 mm cells) and a couple of days, model-independent Mw-evaluation (MStar is great in these cases), and accurate vbar. Don't overspeed.</div>
<div>ad c) cf a), but combine with some EM/AFM and work through the more advanced models in Sedfit/Sedphat. Or try to separate by SEC, etc. Or you know that you have a very narrow mass distribution from agarose gels, etc. (That would make things rather easy.)</div>
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<div>When using CsCl2: thorough, thorough dialysis, vbar measurements and very accurate concentration measurements. Without CsCL2, sufficient ionic strength, as long as that doesn't alter the conformation of the DNA (control with CD?). The mass difference CsCL2 and DNA is so huge in your case, there shouldn't be problems with gradients from CsCL2.</div>
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<div>Good luck!</div>
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<div>Holger</div>
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<div style="margin:0 0 10px 0;"><b>Gesendet:</b> Freitag, 07. Juni 2019 um 03:00 Uhr<br/>
<b>Von:</b> "John Sumida" <jpsumida@uw.edu><br/>
<b>An:</b> "'Lake Paul'" <lake.n.paul@gmail.com><br/>
<b>Cc:</b> 'RASMB' <rasmb@list.rasmb.org><br/>
<b>Betreff:</b> Re: [RASMB] AUC measurements in CsCl?</div>
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<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);">Hi Lake,</span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);"> </span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);">Thank you for taking the time to respond to my question.</span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);"> </span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);">John Sumida</span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);">Molecular Analysis Facility</span></p>
<p class="MsoNormal"><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;color: rgb(31,73,125);">University of Washington</span></p>
<p class="MsoNormal"><b><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;">From:</span></b><span style="font-size: 11.0pt;font-family: Calibri , sans-serif;"> Lake Paul [mailto:lake.n.paul@gmail.com]<br/>
<b>Sent:</b> Thursday, June 06, 2019 3:56 PM<br/>
<b>To:</b> John Sumida <jpsumida@uw.edu><br/>
<b>Cc:</b> RASMB <rasmb@list.rasmb.org><br/>
<b>Subject:</b> Re: [RASMB] AUC measurements in CsCl?</span></p>
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<p class="MsoNormal">John,</p>
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<p class="MsoNormal">To answer your first question, CsCl is not needed to determine the MW of your DNA. Just make sure have the appropriate ionic strength in solution. You will be spinning this slowly, e.g. 10000 rpm or lower. One sample at a time and scan as fast as possible.</p>
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<p class="MsoNormal">Equilibrium on such a large particle is going to take literally forever. I would not do an equilibrium experiment on a 10 MDa particle.</p>
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<p class="MsoNormal">The SV experiment should suffice to get a pretty good molecular weight.</p>
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<p class="MsoNormal">Lake</p>
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<p class="MsoNormal">On Thu, Jun 6, 2019, 5:51 PM John Sumida <<a href="mailto:jpsumida@uw.edu" onclick="parent.window.location.href='mailto:jpsumida@uw.edu'; return false;" target="_blank">jpsumida@uw.edu</a>> wrote:</p>
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<p class="MsoNormal">Dear RASMB,</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">First I have to thank all those who curate this list server as over the last 10 years as a manager of the lab I run, it has and continues to prove a hugely valuable resource for me and the people I serve.</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">My question today has to do with the analysis of DNA and it stems from a user interest in performing an AUC equilibrium experiment in the presence of CsCl. My experience to date has been with nanomaterials and proteins so DNA is a new material for me, however I am under the impression that running AUC samples in presence of CsCl is problematical because of the gradient CsCl itself will generate. </p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">The estimated mass of the species of interest is in the range of 10 mega Daltons; this is also a size regime that is new to me.</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">The objective of the experiment is to characterize nucleic acids extracted from cells which will have a label increasing the mass of the nucleic acid depending on the number of times it has been replicated.</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">Two questions:</p>
<p class="MsoNormal">Is CsCl necessary to determine the size distribution of molecular species >10MDa?</p>
<p class="MsoNormal">Can a CsCl experiment be performed using equilibrium measurements?</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal">Thank you for considering my question.</p>
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<p class="MsoNormal">Best regards,</p>
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<p class="MsoNormal">John Sumida</p>
<p class="MsoNormal">Molecular Analysis Facility</p>
<p class="MsoNormal">University of Washington</p>
<p class="MsoNormal"> </p>
<p class="MsoNormal"><span style="font-size: 10.5pt;font-family: Helvetica , sans-serif;color: rgb(51,51,51);background: white;">In many spheres of human endeavor, from science to business to education to economic policy, good decisions depend on good measurement.</span></p>
<p class="MsoNormal" style="margin-bottom: 12.0pt;"><span style="font-size: 10.5pt;font-family: Helvetica , sans-serif;color: rgb(51,51,51);background: white;">Ben Bernanke</span></p>
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